CN115261287A - Composite lactobacillus microbial inoculum and application thereof - Google Patents
Composite lactobacillus microbial inoculum and application thereof Download PDFInfo
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- CN115261287A CN115261287A CN202211107525.6A CN202211107525A CN115261287A CN 115261287 A CN115261287 A CN 115261287A CN 202211107525 A CN202211107525 A CN 202211107525A CN 115261287 A CN115261287 A CN 115261287A
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- lactobacillus
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- salivarius
- chicks
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
- A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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- A—HUMAN NECESSITIES
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- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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Abstract
The invention discloses a composite lactobacillus microbial inoculum and application thereof. The composite lactobacillus agent is prepared by mixing lactobacillus plantarum freeze-dried powder, lactobacillus acidophilus freeze-dried powder, lactobacillus salivarius freeze-dried powder and lactobacillus reuteri freeze-dried powder according to the bacterial quantity ratio of 1. The four strains in the composite lactobacillus microbial inoculum are safe and reliable, and the fed chicks are added when water is drunk in the chicks stage, so that the chicks can be quickly colonized in vivo in an occupying manner, the disease resistance of the chicks is improved, the immunity of the chicks is enhanced, the intestinal flora balance can be adjusted, the serum immunity index is improved, the intestinal villus development is promoted, and the growth performance of the broilers is improved. Therefore, the composite lactobacillus inoculant can be used as an avian starter product to be safely used in the chick stage, the breeding benefit is improved, and green antibiotic-free breeding is really achieved.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a composite lactobacillus microbial inoculum and application thereof.
Background
The young stage (0-7 days old) of the poultry is the key of the whole breeding success or failure, so that whether the poultry opening products are selected correctly or not is particularly important. The poultry starter product is mainly added under three conditions of an incubator, a transportation process and 7 days before a farm after poultry are shelved. The products aiming at poultry openings in the market at present are mainly divided into two categories, one category is antibiotics mainly comprising cef and ofloxacin, and plant extracts are added into some products, so that the products can quickly kill various pathogenic bacteria vertically propagated in bodies of chicks, reduce the morbidity to a certain extent and improve the survival rate, but the antibiotics damage intestinal tracts of the chicks while killing the pathogenic bacteria and easily cause antibiotic residues and drug-resistant bacteria. The other kind is a nutritional supplement containing multivitamin, various amino acids and saccharides, which can relieve immunity and transportation stress, regulate the low level of maternal antibodies, promote the absorption of nutrients by chicks, increase daily gain and development uniformity, but has the defect that the harm of various pathogenic bacteria cannot be prevented.
The young stage of the bird is also a key stage of the development of the intestinal flora, and the intake and the contact of the probiotics in the stage can influence the establishment of the intestinal flora of the bird. Several species of lactobacillus may colonize early in the growth of avians and are present in the avian animal's crop, anterior part of the digestive tract. The lactobacillus can fast colonize in poultry bodies, so that the poultry has better immunity, and a good foundation is laid for healthy growth in the future.
According to research, the effect of the composite probiotics is often better than that of single probiotics. Therefore, a novel poultry starter product capable of replacing antibiotics and nutritional supplements is developed and can effectively protect the whole breeding cycle of poultry.
Disclosure of Invention
The invention provides a composite lactobacillus microbial inoculum and application thereof. The composite lactobacillus agent comprises four kinds of lactobacillus, namely lactobacillus plantarum, lactobacillus acidophilus, lactobacillus salivarius and lactobacillus reuteri, is beneficial to establishing animal early flora, improves the disease resistance and the immunity of animals, and further improves the production performance of poultry.
In order to achieve the purpose of the invention, the invention is realized by the following technical scheme:
the invention provides a composite lactobacillus preparation which comprises lactobacillus plantarum, lactobacillus acidophilus, lactobacillus salivarius and lactobacillus reuteri.
Further, the composite lactobacillus microbial inoculum is prepared by mixing lactobacillus plantarum lyophilized powder, lactobacillus acidophilus lyophilized powder, lactobacillus salivarius lyophilized powder and lactobacillus reuteri lyophilized powder in a bacterial quantity ratio of 1-3.
Furthermore, the bacterial amount of lactobacillus plantarum, lactobacillus acidophilus, lactobacillus salivarius and lactobacillus reuteri in the composite lactobacillus microbial inoculum is 3000-6000 hundred million CFU/g.
Furthermore, the lactobacillus plantarum freeze-dried powder is prepared by fermenting and drying lactobacillus plantarum GBW-LP001 with the preservation number of CGMCC No.22078.
Further, the lactobacillus acidophilus freeze-dried powder is prepared by fermenting and drying lactobacillus acidophilus L100 with the preservation number of CGMCC No. 10701; the lactobacillus salivarius freeze-dried powder is prepared by fermenting and drying lactobacillus salivarius XJP2 with the preservation number of CGMCC No.11386.
The invention also provides application of the composite lactobacillus microbial inoculum in improving poultry immunity as poultry starter products.
Furthermore, the compound lactobacillus microbial inoculum is added in drinking water in an adding amount of 20 hundred million/day to 200 hundred million/day.
Further, the using time of the composite lactobacillus inoculant is in the young stage of the poultry, and the using time is 6-10 days.
Furthermore, the composite lactobacillus inoculant can improve the survival rate, the average weight, the feed-meat ratio and the serum immunity index of poultry, inhibit the growth of harmful bacteria and increase the total bacteria number in intestinal tracts of poultry.
The invention also provides application of the composite lactobacillus preparation in preparation of a young bird growth promoter.
Compared with the prior art, the invention has the following advantages and technical effects:
1. four lactobacilli in the composite lactobacillus microbial inoculum are all from intestinal tracts of healthy broiler chickens, and antibiotic susceptibility tests prove that the four strains are safe and reliable and meet the safety requirements of direct feeding strains. In the production aspect, the four lactobacillus strains grow faster, the requirement on culture medium is not high, and the method can be used for industrial large-scale production, wherein the acid yield of the lactobacillus plantarum GBW-LP001 is up to 18.1g/L, the four lactobacillus fermentation liquids have good bacteriostatic effects, and the method has good inhibitory effects on pathogenic bacteria such as escherichia coli, staphylococcus aureus, clostridium welchii and the like.
2. The lactobacillus salivarius and lactobacillus reuteri in the composite lactobacillus agent have strong adhesion and colonization capabilities, and can be quickly colonized in intestinal tracts of animals in an occupying mode; the lactobacillus acidophilus in the lactobacillus compositus is beneficial to improving the immunity of young animals, and is added in the early feeding stage of the broiler chickens to facilitate the success of the whole breeding cycle; the lactobacillus plantarum in the composite lactobacillus grows fast, has strong acid production capacity and strong pathogenic bacteria inhibition capacity, and can reduce diseases in the chick stage. Therefore, the composite lactobacillus microbial inoculum is beneficial to establishing early flora of animals, improving the disease resistance and the immunity of the animals and further improving the production performance of the broiler chickens.
3. The composite lactobacillus microbial inoculum provided by the invention is used as a poultry starter product to feed chicks with drinking water, and animal breeding experiments prove that the composite lactobacillus microbial inoculum can regulate the balance of intestinal flora of the broilers, promote the development of intestinal villi, improve the disease resistance and immunity of the broilers and improve the production performance of the broilers.
Drawings
FIG. 1 shows the colony morphology of Lactobacillus acidophilus L100 and Lactobacillus reuteri on plates.
FIG. 2 shows the MIC results of Lactobacillus plantarum GBW-LP001 strain for some antibiotics.
FIG. 3 shows villus development in a portion of the intestine as in example 5.
Fig. 4 is a plot of caecum chyme in different groups from example 5, case two.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to the following specific examples.
In the following examples, unless otherwise specified, the test methods used were all conventional methods, and materials, reagents and the like used were all available from biological or chemical reagents companies.
The formulations of the media required in the following examples are as follows:
1. MRS solid medium: 10.0g/L of peptone, 5.0g/L of beef extract powder, 4.0g/L of yeast extract powder, 20.0g/L of glucose, 2.0g/L of dipotassium phosphate, 2.0g/L of triammonium citrate, 5.0g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.05g/L of manganese sulfate, 15.0g/L of agar, 1.0g/L of Tween 80, and the pH value of the mixture is 6.5 +/-0.2 (25 ℃).
2. MRS liquid medium: 10.0g/L of peptone, 5.0g/L of beef extract powder, 4.0g/L of yeast extract powder, 20.0g/L of glucose, 2.0g/L of dipotassium phosphate, 2.0g/L of triammonium citrate, 5.0g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.05g/L of manganese sulfate, 1.0g/L of tween 80, and 0.5g/L of L-cysteine hydrochloride at the pH value of 6.5 +/-0.2 (25 ℃).
The above culture medium is sterilized at 116 deg.C for 30min before use, and then stored at room temperature.
Example 1: drug susceptibility evaluation of four Lactobacillus strains
In the invention, the lactobacillus acidophilus L100 is derived from intestinal tracts of healthy broilers, has strong acid resistance and slow growth, and is subjected to anaerobic culture for 48 hours at 37 ℃ on an MRS culture medium plate to form small, reticular, rough-surface and irregular-edge colonies (figure 1, left). Lactobacillus reuteri strains grow faster and form larger colonies with smooth surface and regular edges (right in FIG. 1) after being cultured for 48h at 37 ℃ on an MRS medium plate.
The Lactobacillus salivarius XJP2 is preserved in China general microbiological culture Collection center (CGMCC); address: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is as follows: 9/16/2015; the preservation number of the Lactobacillus salivarius is CGMCC No.11386.
The lactobacillus acidophilus L100 is preserved in China general microbiological culture Collection center (CGMCC); address: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is as follows: year 2015, 4 months and 9 days; the preservation number of the Lactobacillus acidophilus is CGMCC No.10701.
The lactobacillus plantarum GBW-LP001 is preserved in China general microbiological culture Collection center (CGMCC); address: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is as follows: 26/03/2021; the preservation number of the Lactobacillus plantarum is CGMCC No.22078.
Lactobacillus reuteri is a commercially available product.
Four kinds of lactobacillus (Lactobacillus plantarum GBW-LP001, lactobacillus acidophilus L100, lactobacillus salivarius XJP2, and Lactobacillus reuteri) were inoculated into MRS liquid medium, cultured overnight at 37 ℃, and then the broth was diluted to 0.5M. And dipping diluted bacteria liquid by using a sterile cotton swab, extruding the swab at the inner wall of the test tube above the liquid surface to remove redundant bacteria liquid, repeatedly and uniformly coating the surface of the prepared MRS solid flat plate for 3 times, rotating the flat plate for 60 degrees every time, and finally coating a circle along the inner source of the flat plate to ensure uniform coating. The antibiotic drug sensitive strip is purchased from AnTu biology.
The four lactobacilli were evaluated for susceptibility to six antibiotics. As shown in table 1 and fig. 2, it was found that four lactobacillus strains were very sensitive to ampicillin, clindamycin and erythromycin, with Minimum Inhibitory Concentration (MIC) values all lower, well below the critical value, MIC values for gentamicin higher than others, and lactobacillus acidophilus L100 reached 16, just above the critical value (table 1 and table 2). Drug sensitivity evaluation shows that the four lactobacillus strains are safe and can be used in animals without worry.
Table 1: MIC results (μ g/mL) for four Lactobacillus strains for six antibiotics
Table 2: critical value (μ g/mL) for different drugs for different lactobacilli
Example 2: growth and acid production evaluation of four lactobacillus strains
Inoculating single colony of four kinds of lactobacillus (Lactobacillus plantarum GBW-LP001, lactobacillus acidophilus L100, lactobacillus salivarius XJP2 and Lactobacillus reuteri) into MRS liquid culture medium, culturing at 37 deg.C overnight, and measuring bacterial liquid OD 600 Then, the bacterial solution OD is added 600 Adjusting to consistent level, inoculating to fresh MRS liquid culture medium at 1%, standing at 37 deg.C for 24 hr, and measuring OD 600 pH and amount of lactic acid produced. Wherein OD 600 Spectral light for valueAnd measuring, wherein the pH value is detected by a pH meter, and the amount of the produced lactic acid is detected by a lactic acid (LD) test box built in Nanjing.
As shown in Table 3, after the strain was cultured and grown at 37 ℃ for 24 hours, the pH of each of the four strains was reduced to 4.35 or less, and the OD of the bacterial liquid was determined 600 The number of the strains is more than 4.60, which indicates that the four strains grow well and are easy to culture. Wherein, the two strains of the lactobacillus plantarum GBW-LP001 and the lactobacillus salivarius XJP2 grow faster because the two strains are homotype fermentation lactic acid bacteria; the lactobacillus acidophilus L100 and the lactobacillus reuteri grow slightly slowly, the lactobacillus reuteri is obligate heterotypic fermentation lactobacillus and has low lactic acid production amount, and the lactobacillus acidophilus is homotypic fermentation, but the strain grows slowly, and the lactic acid production amount is lower than that of the lactobacillus plantarum and the lactobacillus salivarius.
Table 3: growth and acid production of four lactobacillus strains
Example 3: evaluation of bacteriostatic Properties of four Lactobacilli
(1) Culturing the beneficial bacterium liquid: the single colonies of lactobacillus plantarum GBW-LP001, lactobacillus acidophilus L100, lactobacillus salivarius XJP2 and lactobacillus reuteri are respectively inoculated into an MRS liquid culture medium, cultured overnight at 37 ℃, and the bacterial liquid is taken for standby.
(2) And (3) harmful bacterium culture: the four harmful bacteria are Escherichia coli 25922, escherichia coli K88, staphylococcus aureus 25923 and Clostridium welchii ATCC13124. Preparing LB and RCM liquid culture medium in advance, inoculating Escherichia coli 25922, escherichia coli K88 and Staphylococcus aureus 25923 into glycerol tube, and culturing at 37 deg.C and 180rpm for about 20 hr. The clostridium welchii ATCC13124 streaks out a single colony on the RCM culture medium in advance, then inoculates the single colony to the RCM liquid culture medium, and stands for anaerobic culture for about 20h at 37 ℃.
(3) And (3) bacteriostatic test: detection of harmful bacteria OD with spectrophotometer 600 OD of dilution control 600 Adding into sterilized solid culture medium at 50 deg.C by 1% adding amount of 0.3-0.4, mixing, and pouring into a dishAnd after the solidification is completed, punching the hole by using a puncher. Adding 50 mu L of different probiotic bacteria liquid into the holes, then placing the holes at 37 ℃ for culturing for 24h, observing the size of the inhibition zone, and determining the diameter statistical result of the inhibition zone. ( Note: the Escherichia coli uses LB culture medium, clostridium welchii uses RCM culture medium, clostridium welchii is strict anaerobe, need anaerobic culture. The punch diameter was 8mm. )
The test results are shown in table 3: the four strains of lactobacillus have bacteriostasis to four harmful bacteria, wherein the lactobacillus plantarum has the best bacteriostasis effect, the bacteriostasis diameter to staphylococcus aureus 25923 is as high as 19.0mm, the bacteriostasis diameter to clostridium welchii ATCC13124 is 15.0mm, and the bacteriostasis diameters to escherichia coli 25922 and K88 are 14.5mm and 13.5mm respectively. In addition, the effect of the lactobacillus reuteri on inhibiting escherichia coli is also prominent, and the effect is probably related to the produced reuterin.
Table 3: bacteriostatic results of four strains of Lactobacillus (bacteriostatic diameter, unit mm)
Example 4: fermentation of four lactobacillus strains and preparation of composite microbial inoculum
Laboratory shake flask fermentation of four strains of lactobacillus (Lactobacillus plantarum GBW-LP001, lactobacillus acidophilus L100, lactobacillus salivarius XJP2 and Lactobacillus reuteri) with a fermentation bacteria amount of 20-30 hundred million CFU/mL.
Then carrying out small tank fermentation of 50L, wherein the fermentation conditions are as follows: the inoculation amount is 2%, the culture temperature is 37 ℃, the rotating speed is 150rpm, ventilation is not performed, the tank pressure is 0.5Mpa, the pH value of ammonia water in the process is controlled to be 6.50, and the culture period is 12-16h; the amount of four lactobacillus ferments is above 140 hundred million CFU/mL, wherein the amount of lactobacillus plantarum GBW-LP001 is 200 hundred million CFU/mL, the amount of lactobacillus acidophilus L100 is 140 hundred million CFU/mL, the amount of lactobacillus salivarius XJP2 is 179 hundred million CFU/mL, and the amount of lactobacillus reuteri is 160 hundred million CFU/mL.
The formula of the small-tank fermentation medium is as follows: 30-38g/L of glucose, 12-18g/L of sucrose, 14-20g/L of peptone, 10-18g/L of yeast extract, 13-18g/L of beef extract, 5-10g/L of sodium acetate, 0.5-1.0g/L of dipotassium hydrogen phosphate, 0.5-1.0g/L of potassium dihydrogen phosphate, 1.2-1.8g/L of triammonium citrate, 0.5-1.0g/L of magnesium sulfate, 0.1-0.5g/L of manganese sulfate, 1-5g/L of Tween, and 0.2-0.8g/L of L-cysteine hydrochloride.
After being fermented in a 50L small tank, four strains of lactobacillus fermentation liquor are frozen and dried to obtain freeze-dried powder with 3000-6000 hundred million CFU/g.
And mixing the four lactobacillus powder according to the same bacterial quantity to obtain the composite lactobacillus preparation product.
Example 5: application evaluation of composite lactobacillus on broiler chicken
Case one, influence of composite lactobacillus on growth of broiler chicken in opening period
Test site: animal house of Qingdao root biology flatness base
Test materials: composite lactobacillus product 1 (mixed with four bacteria), composite lactobacillus product 2 (mixed with lactobacillus plantarum GBW-LP001 and lactobacillus salivarius XJP 2), composite lactobacillus product 3 (mixed with lactobacillus acidophilus L100 and lactobacillus reuteri)
And (3) experimental design:
table 4: case-test design
Selecting 500 feathers of high-quality white feather broilers of 1 day old, screening out chickens with obvious weight difference, and dividing the chickens with the residual 450 feathers into 18 columns (25 chickens in each column) to require that the average weight of the chickens in each column is approximate.
The test was divided into 5 treatment groups, 4 columns for control group, test group 1 and test group 2, and 3 columns for test group 3 and test group 4 (note: the columns of the control group and the test group are arranged alternately).
The test period is 42 days, 1-7 days, the test group is fed with the lactobacillus complex, and is not fed for 8-42 days. The granular materials are taken to freely drink water, and artificial illumination is adopted to immunize Newcastle disease and infectious bronchitis vaccines. Antibiotics are not used as growth promoters in the whole process. Growth performance indexes are recorded every week in the test process, 8 chickens are randomly selected from each group at the age of 42 days, and cecal chyme and jejunum middle section are taken.
Index measurement: (1) microorganism diversity index: extracting total DNA of the excrement from the caecum chyme, and detecting the total bacteria number, the lactobacillus number and the escherichia coli number by using a fluorescent quantitative PCR instrument through a fluorescent quantitative kit;
(2) VFA index: detecting the VFA content of the intestinal chyme by using a gas chromatograph;
(3) Slicing jejunum: the midjejunum is taken and soaked in paraformaldehyde solution for paraffin embedding, HE staining and scanning by Beijing Zhongke Wanbang Biotech Co.
The obtained jejunal section is shown in fig. 3, the test results are shown in tables 5 to 9, and the results of various indexes are as follows:
the production performance is as follows: compared with a control group, the survival rate, the average weight and the feed meat ratio of each group of the test group are superior, but no significant difference exists at the age of 7 days, and compared with the control group and other test groups, the survival rate, the average weight and the feed meat ratio of the test group 2 are superior and significantly different at the age of 42 days;
in terms of microbial diversity: compared with the control group, each group of the test group can reduce the number of escherichia coli, increase the total bacteria number and the lactobacillus number, and the test group 1 is most prominent in reducing the escherichia coli;
VFA aspect: the test groups were all higher in acetic acid, butyric acid, total VFA than the control group, test group 2 performed best on acetic acid and branched VFA/total VFA, whereas test group 1 was the highest in butyric acid content, presumably related to test group 1 in reducing the number of escherichia coli;
in the aspect of villus development: the groups were better in villus length, crypt depth, and villus hiding ratio than the control group, and test group 2 was better.
The above test results demonstrate that:
(1) The addition of the lactobacillus complex in the early growth stage of the broiler chickens is critical to the whole growth cycle;
(2) The effect of the composite lactobacillus product 1 is better than 200 hundred million/one/day according to the addition amount of 100 hundred million/one/day;
(3) The effect of the composite lactobacillus product 1 (the composite of four kinds of lactobacillus) is better than that of the composite lactobacillus product 2 and the composite lactobacillus product 3;
(4) More species of lactobacillus complex is better, but not more is better;
(5) The high-addition-amount composite lactobacillus does no harm to the growth of the broilers.
Table 5: statistics of 7 days old production performance
Table 6: statistics of 42 days old production performance
Table 7: analysis of microbial diversity (Lg value)
Table 8: VFA analysis of cecal chyme
Table 9: intestinal villus height and crypt depth analysis
Case two, influence of lactobacillus compositus on immunity of broiler chicken
Test site: animal house of Qingdao root biological flatness base
Test materials: compound lactobacillus product (four kinds of bacteria mixed)
And (3) experimental design:
table 10: case two test design
Selecting 500 feathers of high-quality white feather broilers of 1 day age, screening out chickens with obvious weight difference, dividing the chickens with the remaining 450 feathers into 18 columns (25 chickens in each column), and requiring that the average weight of the chickens in each column is approximate.
The test was divided into 3 treatment groups with 6 fields per treatment group (note: the control and test group fields are arranged across each other).
Continuously adding the vaccine every day at 14d,1-3 days of age, continuously adding the vaccine every day at 6-8 days of age, not adding the vaccine in the rest time, feeding granular feed for freely drinking water, and adopting artificial illumination to immunize new strains at 1 day of age (infectious bronchitis live vaccine) and immunize new strains at 7 days of age (Newcastle disease and infectious bronchitis bigeminal live vaccine). Antibiotics are not used as growth promoters in the whole process.
Sampling and index determination: at 14 days of age, 8 chickens were randomly selected from each group, and cecal chyme and blood were collected. The indexes are determined as follows:
(1) And (3) production performance statistics: counting the survival rate, the average weight and the feed conversion ratio every week;
(2) Microorganism diversity index: extracting total DNA of the excrement from the caecum chyme, and quantitatively detecting the total bacteria number, the lactobacillus number and the escherichia coli number by fluorescence;
(3) Serum index: igG and glutathione peroxidase (GSH-Px) were detected using an ELISA kit for IgG and GSH-Px.
During the sampling process of the killed chickens at the age of 14 days, the chyme amount of the control group is obviously less than that of the test group, and the color of the chyme is slightly lighter than that of the test group, as shown in figure 4, which is related to the improvement of the feed intake of the animals after the feeding of the lactobacillus compositus.
The test results are shown in tables 11 to 14, specifically as follows:
the production performance is as follows: the survival rate, the average weight and the feed conversion ratio of the test group are better than those of the control group, the test group 2 is optimal, and the advantage of 14-day-old is more obvious than that of 7-day-old;
microbial diversity: compared with the control group, the two test groups have slight advantages in the total bacteria, the lactobacillus number and the escherichia coli number, and only the test group 1 has obvious difference in the escherichia coli number from the control group;
serum immunity index: the IgG and GSH-Px indexes of the two test groups are higher than those of the control group, and the difference between the test group 2 and the control group is very obvious.
And (4) test conclusion: in the chick stage and under the stress condition, the feeding of the lactobacillus complex is helpful for improving the immunity of the broilers and improving the production performance of the broilers, but the more the addition amount is, the better the addition amount is.
Table 11: statistics of 7 days old production performance
Table 12:14 day old production performance statistics
Table 13: analysis of microbial diversity (Lg value)
Table 14: serum immune index analysis
The above examples are only intended to illustrate the technical solution of the present invention, and not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described in the foregoing embodiments, or equivalents may be substituted for some of the features thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (10)
1. A composite lactobacillus preparation is characterized by comprising lactobacillus plantarum, lactobacillus acidophilus, lactobacillus salivarius and lactobacillus reuteri.
2. The composite lactobacillus agent of claim 1, wherein the composite lactobacillus agent is prepared by mixing lactobacillus plantarum lyophilized powder, lactobacillus acidophilus lyophilized powder, lactobacillus salivarius lyophilized powder and lactobacillus reuteri lyophilized powder in a ratio of 1.
3. The complex lactobacillus preparation according to claim 1, wherein the amounts of lactobacillus plantarum, lactobacillus acidophilus, lactobacillus salivarius and lactobacillus reuteri are all 3000-6000 hundred million CFU/g.
4. The composite lactobacillus agent as claimed in claim 2, wherein the lactobacillus plantarum lyophilized powder is prepared by fermenting and drying lactobacillus plantarum GBW-LP001 with the preservation number of CGMCC No.22078.
5. The composite lactobacillus agent as claimed in claim 2, wherein the freeze-dried lactobacillus acidophilus powder is prepared by fermenting and drying lactobacillus acidophilus L100 with the preservation number of CGMCC No. 10701; the lactobacillus salivarius freeze-dried powder is prepared by fermenting and drying lactobacillus salivarius XJP2 with the preservation number of CGMCC No.11386.
6. Use of the complex lactobacillus agent of any of claims 1 to 5 for enhancing immunity in poultry as a poultry starter product.
7. The use of claim 6, wherein the complex lactobacillus preparation is added as drinking water in an amount of 20 hundred million/day to 200 hundred million/day.
8. The use of claim 6, wherein the lactobacillus complex inoculant is used for the young stage of poultry for 6-10 days.
9. The use of claim 6, wherein the lactobacillus complex inoculant is capable of improving the survival rate, average weight, feed-meat ratio and serum immunity index of poultry, inhibiting the growth of harmful bacteria, and increasing the total bacteria number in intestinal tracts of poultry.
10. The use of the complex lactobacillus inoculant of any one of claims 1-5 in the preparation of a chick growth promoter.
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Application publication date: 20221101 |